Hydrogel composition for cell culture apparatus

ABSTRACT

A cell culture apparatus and a method for fabricating the cell culture apparatus are disclosed, the method comprises forming at least one fillister on a biomaterial composite layer by photolithography, wherein the biomaterial composite layer contains two gel materials. One is a bio-compatible hydrogel composition having various weight ratio of: 2-hydroxyethylmathacrylate (HEMA), bisphenolA and glycidyl methacrylate (bis-GMA), triethylene glycol dimethacrylate (TEGDMA), r-methacryloxypropyl trimethoxysilane (MAPTMS), α,α-diethoxyacetophenone (DEAP), and the other one is a photo-sensitive silica gel composition.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a cell culture apparatus and thefabricating method of the same, especially to a cell culture apparatusand its fabricating method for cell culturing.

2. Description of Related Art

Conventional cell culture apparatus for in vitro cell culture orbioreactors in laboratories has certain limits in culturing cells formedical use. The shortcoming of the conventional cell culture apparatusfor in vitro cell culture cannot show the real activity of a cell invivo because of the conventional cell culture apparatus lacks masstransfer system. On the other hand, the conventional bioreactor issuitable for protein drugs only. Besides, the bioreactor occupies largespace and needs complicated operating process. Furthermore, the shearstrength of fluid, and the micro-environment lack of physical similarityin the system of the bioreactor makes tissue specific cells withbiological functions not be cultured. For example, if the isolated livercells are cultured without other kinds of cells in the bioreactor, theliver cells will lose their mature physical function in one week, andthe liver cells will be dead eventually.

Recently, several systems for culturing cells in vitro but with realactivity in vivo, such as Extracellular Matrix (ECM), Media modificationand co-culture system, are proposed. The bio-compatible polymer of aconventional cell culture frame (cell culture platform) used in themethods mentioned above are produced by sol-gel method. The pore size ofthe frame made of the sol-gel method is in the range of 50-100 μm.However, the size of a normal cell is in the range of 5-20 μm. Owing tothe pore is bigger than the size of the cells, cell clusters will beformed in the pore of the frame. Moreover, the pore size of a cellculture frame produced by conventional methods can not be narrow down to100 μm or less. And the precise size of the structure is not easy to bere-produced. Hence the systems for culturing cells in vitro illustratedabove cannot be used for mass production.

Therefore, it is desirable to provide an improved method to mitigate theaforementioned problems.

SUMMARY OF THE INVENTION

A hydrogel composition, comprising: 50-100 wt % of 2-hydroxyethylmathacrylate (HEMA); 5-30 wt % of triethylene glycol dimethacrylate(TEGDMA); 5-30 wt % of r-methacryloxypropyl trimethoxysilane (MAPT MS);and 5-15 wt % of α,α-diethoxyacetophenone (DEAP).

The hydrogel composition of the present invention further comprising5-50 wt % of bisphenol A and glycidyl methacrylate (Bis-GMA).

Another aspect of the present invention is related to a cell cultureapparatus comprising: a substrate; and at least one layer ofbio-compatible material forming on said substrate, wherein the materialis hydrogel composition or photo-sensitive silica gel composition, and apattern with at least one recess is formed on said layer ofbio-compatible material.

Further, a method for fabricating the cell culture apparatus of thepresent invention is also disclosed. The method comprises the steps of:(a) providing a substrate; (b) forming a layer of bio-compatiblehydrogel composition, or photo-sensitive silica gel composition; and (c)forming a pattern of at least one recess on said layer of bio-compatiblematerial by photolithography.

The method for fabricating the cell culture apparatus of the presentinvention, further comprising the steps of: (d) filling said recess witha sacrificed material;

(e) exposing said recess and rinsing (b) forming a layer ofbio-compatible hydrogel composition, or photo-sensitive silica gelcomposition; (c) forming a pattern of at least one recess on said layerof bio-compatible material by photolithography; and (f) removing saidsacrificed material.

Moreover, wherein the steps (d), (e), (b) and (c) can be repeated one tofive times as desired.

The material used for the cell culture apparatus have good biocompatiblefeature. Besides, it is also suitable for manufacturing process ofphotolithography. Therefore, cell culture apparatus with microfluidicchannel can be produced through photolithography in batch through themethod of the present invention. Also, the micro-sized (μm) dimension ofthe cell culture apparatus can be easily controlled. Hence, the cellculture can be re-produced precisely.

Besides, the biocompatible material of the cell culture apparatus hasporous structure. Cultured cells can communicate each other by releasedsignals through the porous water gel between different cell cultureplatforms. The interactions or signal regulations between cellsstabilize physical activities of target cells, therefore their lifespanand biological functions can be prolonged.

The present invention also provides a cell culture apparatus, comprisinga substrate; and a layer of bio-compatible material forming on saidsubstrate, wherein the material is hydrogel composition orphoto-sensitive silica gel composition, and a pattern with at least onerecess on said layer of bio-compatible material.

The cell culture apparatus of the present invention is composed withhydrogel composition, which is optionally comprises: bisphenol A andglycidyl methacrylate (Bis-GMA) (viscous material), triethylene glycoldimethacrylate (TEGDMA) (cross linker), r-methacryloxypropyltrimethoxysilane (MAPT MS) (adhesion improving reagent),α,α-diethoxyacetophenone (DEAP) (photo-sensitive agent) or thecombinations thereof.

The hydrogel composition preferably comprises: 50-100 weight percentageof 2-hydroxyethyl mathacrylate (HEMA), 0-50 weight percentage,preferably 5-50 weight percentage of bisphenol A and glycidylmethacrylate (Bis-GMA), 5-30 weight percentage of triethylene glycoldimethacrylate (TEGDMA), 5-30 weight percentage of r-methacryloxypropyltrimethoxysilane (MAPT MS), and 5-15 weight percentage ofα,α-diethoxyacetophenone (DEAP).

In the method of the present invention, the photolithography of step (b)can comprise any step of conventional photolithography. Preferably, thephotolithography of step (b) comprises exposure and development. Thelight source for photolithography can be any conventional light used forcuring bio-compatible materials. Preferably, the bio-compatible materialis exposed to UV light.

In the method of the present invention, the substrate can be anyconventional substrates, more preferably is made from transparent orsemi-transparent materials. The substrate material is preferablyselected from the group consisting of glass, silicon, plastic, rubber,ceramic and the combination thereof.

In the present invention method, the step of development can beperformed through any conventional process. Preferably, thebio-compatible material is developed by any a developer. And thedeveloper used in the present method can be any solvent used to dissolvebio-compatible materials, e.g., HEMA. Preferably, the solvent isethanol, acetone or the mixture thereof. The recess formed on thebio-compatible material can be of any pattern. Preferably, the recess isat least one microfluidic channel.

The width of the microfluidic channel is preferably in a range from 1 μmto 1000 μm, and more preferably is between 1 μm to 100 μm. Thesacrificed material used in the present method is2-acrylamido-2-methyl-propanesulfonic acid (AMPS),N-isopropyl-acrylamide (NiPAAm) or methacrylic acid (MAA). Preferably,the sacrificed material is AMPS.

The step (d) in the method of the present invention is preferablyachieved by water rinsing. The step (a) is performed by any knownprocess, preferably is by spin coating. The bio-compatible materialexposed to UV light undergoes a photo-curing reaction. Preferably, thephoto-curing reaction is polymerization.

The bio-compatible material is photo-cured to obtain a micro structurethrough a photolithography process, such as UV exposure. The microstructure operates in coordination with the pattern of microfluidicchannels is so-called a cell micro patterning. The main feature of thecell culture apparatus of the present invention is optionally of aporous or non-porous configuration. The main material of the cellculture apparatus depends on the purposes of cell interaction or cellisolation.

A three dimensional scaffold of the cell culture apparatus is obtainedthrough repeated steps of exposure and development. Different cell linescan have different distributions by simply controlling the flow ofmicrofluid, The cell micro patterning in the cell culture apparatus thuscan be obtained. Therefore, the cell culture environment or physicaltissue structure in the present cell culture apparatus is more imitativeto those in vivo by controlling the distribution or combination ofdifferent cells.

Moreover, the water gel composition and the photosensitive siliconcomposition are transparent or semi-transparent materials. Therefore, itis suitable for embedding the culture plate and proceeding tissuesection other than monitoring the lysed bio-molecules.

Furthermore, the cell culture apparatus of the present invention can beconnected or equipped with existing microscope systems directly formonitoring in real time.

Other objects, advantages, and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the flowchart of the fabricating method of the cellculture apparatus of the present embodiment;

FIG. 2 is a diagram illustrating the structure of the cell cultureapparatus in example 1 of the present invention;

FIG. 3 is a flow chart of another embodiment in example 2 of the presentinvention method;

FIG. 4 is the photo of cultured cells in example 1;

FIG. 5 is the photo of cultured cells in example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Example 1

The material of the cell culture apparatus of the present invention is ahigh biocompatible material such as 2-hydroxyethyl mathacrylate (HEMA).The material is optionally combined with viscous material, cross-linker,adhesion improving reagent and photo-sensitive agent. The biocompatiblematerial is photo-cured after exposing to UV light, and the curedmaterial can be adhered well to the substrate of the cell culturesubstrate.

The biocompatible material of water gel composition of the presentinvention comprises: 50-100 weight percentage of 2-hydroxyethylmathacrylate (HEMA), 0-50 weight percentage, preferably 5-50 weightpercentage of phenol A and glycidyl methacrylate (Bis-GMA), 5-30 weightpercentage of triethylene glycol dimethacrylate (TEGDMA), 5-30 weightpercentage of r-methacryloxypropyl trimethoxysilane (MAPT MS), and 5-15weight percentage of α,α-diethoxyacetophenone (DEAP).

The main material of the cell culture apparatus is HEMA. The Bis-GMA isused as viscous material. TEGDMA acts as the cross-linker, MAPTMS is theadhesion improving reagent, and DEAP is the photosensitive agent. Thephotosensitive agent in the HEMA can be cured by polymerization throughUV light exposure. FIG. 1 illustrates the flowchart of the fabricatingmethod of the cell culture apparatus of the present embodiment. First,providing a substrate 1, the substrate material is selected from thegroup consisting of glass, silicon, plastic, rubber, ceramic and thecombination thereof. And the biocompatible adhesion-improving reagent ofthe correlative substrate material is alkoxysilanes, halosilanes,alkylthiols, or alkylphosphsnates. In the present embodiment, thesubstrate material is glass, and the adhesion-improving reagent isMAPTMS.

Second, a layer of biocompatible material is formed on the substrate 1.In the present embodiment, a first material layer 2 is formed on thesubstrate 1 by spin coating. The biocompatible material used in thepresent embodiment comprises: 5 wt % of HEMA, 4 wt % of Bis-GMA, 2.7 wt% of TEGDMA, 2.7 wt % of MAPTMS, and 1.35 wt % of DEAP.

Then, a pattern is formed on the first material layer 2 throughphotolithography. As it is shown in the middle figure of FIG. 1, aphotomask 3 is aligned over the first material layer 2, and exposed tolight. The first material layer 2 exposed to UV light undergoespolymerization and becomes insoluble in the developer. On the contrary,non-exposed portion of the first material layer 2 is not polymerized,meanwhile, the HEMA exists in the form of monomer and it remains solublein the developer.

Finally, non-exposed portion is removed by the developer, and the moldedmicrofluidic channels defined by the photomask is obtained on the firstmaterial layer 2. The light for exposure in the present invention can beany conventional light used for curing the biocompatible material in thepresent invention. The developer can be any conventional solvent toremove uncured biocompatible material in the present invention. In thepresent embodiment, the first material layer 2 is exposed to UV light(365 nm, 100 W/cm²) for 60 seconds with the photomask 3. A developercontaining acetone and ethanol in a ratio of 50:50 is used to removeuncured biocompatible material. A layer of micropattern is formed, andit is used as the platform for cell culturing.

The micropattern of the cell culture apparatus of the present inventionhave at least one recess 21. The forming process of the recess 21 isdifferent with various patterns of photomasks and the process ofphotolithography. The size of micropattern is various by differentphotomasks. In the present embodiment, the micropattern is amicrofluidic channel with a width of 5 μm.

Cells with the same or different phenotypes can be cultured in the samecell culture apparatus of the present invention. As it is shown in FIG.2, an upper cover 4 is formed with a soft material, such as silicon.There forms an inlet hole 41 and an outlet hole 411 on the upper cover 4for cells flowing. Then, the upper cover 4 is contact with the firstmaterial layer 2 tightly, and the inlet hole 41 of the upper cover 4 isconnected to the recess 21 of the first material layer 2. Cells areapplied into to the recess 21 of the first material layer 2 through theinlet hole 41. The cells are retained in the bottom of the recess 21(the surface of the substrate 1). Circulating culture media is thenapplied after the cells attached completely. Similarly, the other recess22 is used for cell culturing by applying cells and culture mediathrough inlet 42 and outlet 422. The cells culture in the recess 21 andrecess 22 can be different or the same.

FIG. 4 shows the photo of cultured cells. The condition for cellculturing is: (1) rinsing the culture plate with micropattern with PBSsolution twice; (2) suspending 5×10⁵ cells/ml of CA 3 cells in MEMculture medium containing 10% FBS, then seeding the cells into theculture plate for one-day culturing; (3) removing the culture medium andrinsing the plate with PBS for twice, for removing un-attached cells ornon-clustered cells. Then, keep monitoring the condition of cellgrowing.

The cell culture apparatus can be used to monitor the lysedbio-molecules. Moreover, it is suitable for embedding the culture plateand proceeding tissue section, because the HEMA composition and thephotosensitive silicon composition are transparent. The biocompatiblematerial used in the present invention is a porous water gel (HEMA). Thebiological signals (e.g. proteins) released from a cell can betransmitted to another surrounding cell through the porousmicrostructure. Therefore, the purpose of co-culturing cells achieves.The problem of low growth rate in culturing isolated liver cells invitro can also be solved. Furthermore, the cell culture apparatus of thepresent invention performs various combinations of cell lines. Itimitates more closely to a real human tissue since the real tissue iscomposed with multiple cell lines.

Another embodiment is shown in FIG. 3. The multiple culture layers canbe prepared following the method described above. A three-dimensionalcell scaffold is formed via spin coating and photolithography. Therecesses 21, 22 on the single layer of cell culture platform (as shownin the bottom figure in FIG. 1) are filled with a sacrificed material 7.Steps of spin coating and photolithography are repeated to form thesecond material layer 5. Then, another photomask 6 is aligned to thesecond material layer 5, and the second material layer 5 is exposed toUV light. Polymerization is introduced in the exposed portion of thesecond material layer 5. The portion of non-exposed second materiallayer 5 is then removed by a developer Thus, a micropattern is formed.In the present embodiment, the micropattern is a microfluidic channel,which has at least one groove with width of 5 μm. Finally, thesacrificed material 7 is removed. And a three dimensional scaffold ofmultiple cell culture platforms with network pattern is created. In thepresent embodiment, the sacrificed material is AMPS, and it is removedby water rinsing.

Example 2

The material of the cell culture apparatus of the present invention canbe a photosensitive silicon composition in place of HEMA composition.

The process is the same as described in example 1. A glass substrate 1is provided as shown in the upper figure of FIG. 1. About 3 ml ofpatternable silicon rubber (Corning, WL-5350) is applied onto the glasssubstrate 1. The substrate is spun on a spin coater in 500 rpm for 30seconds, and a first material layer 2 with 50 μm thickness is formed.Then place the glass substrate on the hot plate for soft baking under110° C.-120° C.

Refer to the middle figure of FIG. 1. The silicon rubber (the firstmaterial layer 2) is exposed to UV light (600-1000 mJ/cm²) in anexposure. The post exposure baking is performed on the glass substrateon the hot plate under 150° C. Subsequently, the cell culture platformis created after developing for one hour by a negative develop reagent.FIG. 5 shows the photo of cultured cell of the present embodiment, andthe condition is the same as described in example 1.

It spends less time in fabricating the cell culture apparatus of thepresent invention. The structure dimension with micro-size can becontrolled precisely, and can be batch re-produced, because themicropattern is formed via photolithography. Besides, since thebiocompatible material HEMA forms a porous water gel, cells cancommunicate each other by released signals through the porous water gelbetween different cell culture platforms.

In the conventional techniques, the materials used for preparingmicropattern via photolithography are PDMS, PEG or other materialswithout biocompatible feature.

The present invention provides a method for fabricating a multiple-usedcell culture apparatus, and it can be produced in large quantity. Theapparatus also provides a microenvironment, which closely imitates anative cell environment for a better monitoring of cell metabolism invitro. In addition, the cell culture apparatus can combine withautomatic systems for high throughput and high content drug candidatescreening.

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

1. A hydrogel composition, comprising: 50-100 wt % of 2-hydroxyethylmathacrylate (HEMA); 5-30 wt % of triethylene glycol dimethacrylate(TEGDMA); 5-30 wt % of r-methacryloxypropyl trimethoxysilane (MAPT MS);and 5-15 wt % of α,α-diethoxyacetophenone (DEAP).
 2. The hydrogelcomposition as claimed in claim 1, further comprising: 5-50 wt % ofbisphenol A and glycidyl methacrylate (Bis-GMA).